Shr3p Mediates Specific COPII Coatomer–Cargo Interactions Required for the Packaging of Amino Acid Permeases Into ER-derived Transport Vesicles

The SHR3 gene of Saccharomyces cerevisiae encodes an integral membrane component of the endoplasmic reticulum (ER) with four membrane-spanning segments and a hydrophilic, cytoplasmically oriented carboxyl-terminal domain. Mutations in SHR3 specifically impede the transport of all 18 members of the amino acid permease (aap) gene family away from the ER. Shr3p does not itself exit the ER. Aaps fully integrate into the ER membrane and fold properly independently of Shr3p. Shr3p physically associates with the general aap Gap1p but not Sec61p, Gal2p, or Pma1p in a complex that can be purified from N-dodecylmaltoside-solubilized membranes. Pulse–chase experiments indicate that the Shr3p–Gap1p association is transient, a reflection of the exit of Gap1p from the ER. The ER-derived vesicle COPII coatomer components Sec13p, Sec23p, Sec24p, and Sec31p but not Sar1p bind Shr3p via interactions with its carboxyl-terminal domain. The mutant shr3-23p, a nonfunctional membrane-associated protein, is unable to associate with aaps but retains the capacity to bind COPII components. The overexpression of either Shr3p or shr3-23p partially suppresses the temperature-sensitive sec12-1 allele. These results are consistent with a model in which Shr3p acts as a packaging chaperone that initiates ER-derived transport vesicle formation in the proximity of aaps by facilitating the membrane association and assembly of COPII coatomer components.

Alternative splicing of the rat sodium/bile acid transporter changes its cellular localization and transport properties

Bile secretion involves the structural and functional interplay of hepatocytes and cholangiocytes, the cells lining the intrahepatic bile ducts. Hepatocytes actively secrete bile acids into the canalicular space and cholangiocytes then transport bile acids in a vectorial manner across their apical and basolateral plasma membranes. The initial step in the transepithelial transport of bile acids across rat cholangiocytes is apical uptake by a Na+-dependent bile acid transporter (ASBT). To date, the molecular basis of the obligate efflux mechanism for extrusion of bile acids across the cholangiocyte basolateral membrane remains unknown. We have identified an exon-2 skipped, alternatively spliced form of ASBT, designated t-ASBT, expressed in rat cholangiocytes, ileum, and kidney. Alternative splicing causes a frameshift that produces a 154-aa protein. Antipeptide antibodies detected the ≈19 kDa t-ASBT polypeptide in rat cholangiocytes, ileum, and kidney. The t-ASBT was specifically localized to the basolateral domain of cholangiocytes. Transport studies in Xenopus oocytes revealed that t-ASBT can function as a bile acid efflux protein. Thus, alternative splicing changes the cellular targeting of ASBT, alters its functional properties, and provides a mechanism for rat cholangiocytes and other bile acid-transporting epithelia to extrude bile acids. Our work represents an example in which a single gene appears to encode via alternative splicing both uptake and obligate efflux carriers in a bile acid-transporting epithelial cell.

Evolutionary specialization of the nuclear targeting apparatus

The α- and β-karyopherins (Kaps), also called importins, mediate the nuclear transport of proteins. All α-Kaps contain a central domain composed of eight approximately 40 amino acid, tandemly arranged, armadillo-like (Arm) repeats. The number and order of these repeats have not changed since the common origin of fungi, plants, and mammals. Phylogenetic analysis suggests that the various α-Kaps fall into two groups, α1 and α2. Whereas animals encode both types, the yeast genome encodes only an α1-Kap. The β-Kaps are characterized by 14–15 tandemly arranged HEAT motifs. We show that the Arm repeats of α-Kaps and the HEAT motifs of β-Kaps are similar, suggesting that the α-Kaps and β-Kaps (and for that matter, all Arm and HEAT repeat-containing proteins) are members of the same protein superfamily. Phylogenetic analysis indicates that there are at least three major groups of β-Kaps, consistent with their proposed cargo specificities. We present a model in which an α-independent β-Kap progenitor gave rise to the α-dependent β-Kaps and the α-Kaps.

Allosteric crosstalk between peptide-binding, transport, and ATP hydrolysis of the ABC transporter TAP

The transporter associated with antigen processing (TAP) is essential for intracellular transport of protein fragments into the endoplasmic reticulum for loading of major histocompatibility complex (MHC) class I molecules. On the cell surface, these peptide–MHC complexes are monitored by cytotoxic T lymphocytes. To study the ATP hydrolysis of TAP, we developed an enrichment and reconstitution procedure, by which we fully restored TAP function in proteoliposomes. A TAP-specific ATPase activity was identified that could be stimulated by peptides and blocked by the herpes simplex virus protein ICP47. Strikingly, the peptide-binding motif of TAP directly correlates with the stimulation of the ATPase activity, demonstrating that the initial peptide-binding step is responsible for TAP selectivity. ATP hydrolysis follows Michaelis–Menten kinetics with a maximal velocity Vmax of 2 μmol/min per mg TAP, corresponding to a turnover number of approximately 5 ATP per second. This turnover rate is sufficient to account for the role of TAP in peptide loading of MHC molecules and the overall process of antigen presentation. Interestingly, sterically restricted peptides that bind but are not transported by TAP do not stimulate ATPase activity. These results point to coordinated dialogue between the peptide-binding site, the nucleotide-binding domain, and the translocation site via conformational changes within the TAP complex.

Characterization of complex mineral assemblages: Implications for contaminant transport and environmental remediation

Surface reactive phases of soils and aquifers, comprised of phyllosilicate and metal oxohydroxide minerals along with humic substances, play a critical role in the regulation of contaminant fate and transport. Much of our knowledge concerning contaminant-mineral interactions at the molecular level, however, is derived from extensive experimentation on model mineral systems. Although these investigations have provided a foundation for understanding reactive surface functional groups on individual mineral phases, the information cannot be readily extrapolated to complex mineral assemblages in natural systems. Recent studies have elucidated the role of less abundant mineral and organic substrates as important surface chemical modifiers and have demonstrated complex coupling of reactivity between permanent-charge phyllosilicates and variable-charge Fe-oxohydroxide phases. Surface chemical modifiers were observed to control colloid generation and transport processes in surface and subsurface environments as well as the transport of solutes and ionic tracers. The surface charging mechanisms operative in the complex mineral assemblages cannot be predicted based on bulk mineralogy or by considering surface reactivity of less abundant mineral phases based on results from model systems. The fragile nature of mineral assemblages isolated from natural systems requires novel techniques and experimental approaches for investigating their surface chemistry and reactivity free of artifacts. A complete understanding of the surface chemistry of complex mineral assemblages is prerequisite to accurately assessing environmental and human health risks of contaminants or in designing environmentally sound, cost-effective chemical and biological remediation strategies.

Hexose Transport in Growing Petunia Pollen Tubes and Characterization of a Pollen-Specific, Putative Monosaccharide Transporter1

We investigated the molecular and physiological processes of sugar uptake and metabolism during pollen tube growth and plant fertilization. In vitro germination assays showed that petunia (Petunia hybrida) pollen can germinate and grow not only in medium containing sucrose (Suc) as a carbon source, but also in medium containing the monosaccharides glucose (Glc) or fructose (Fru). Furthermore, high-performance liquid chromatography analysis demonstrated a rapid and complete conversion of Suc into equimolar amounts of Glc and Fru when pollen was cultured in a medium containing 2% Suc. This indicates the presence of wall-bound invertase activity and uptake of sugars in the form of monosaccharides by the growing pollen tube. A cDNA designated pmt1 (petunia monosaccharide transporter 1), which is highly homologous to plant monosaccharide transporters, was isolated from petunia. Pmt1 belongs to a small gene family and is expressed specifically in the male gametophyte, but not in any other vegetative or floral tissues. Pmt1 is activated after the first pollen mitosis, and high levels of mRNA accumulate in mature and germinating pollen. A model describing the transport of sugars to the style, the conversion of Suc into Glc and Fru, and the active uptake by a monosaccharide transporter into the pollen tube is presented.

Expression of Small Heat-Shock Proteins at Low Temperatures1 : A Possible Role in Protecting against Chilling Injuries

We previously reported that short exposure of tomato (Lycopersicon esculentum L.) fruits to high temperature protects them from chilling injury. To study the involvement of heat-shock proteins (HSPs) in the acquisition of low-temperature tolerance, we cloned two heat-shock-induced genes that are also expressed at low temperatures. The cloned cDNAs belong to the small HSP group. Sequence analyses of the clones showed perfect homology to the tomato-ripening gene tom66 and to the tomato chloroplastic HSP21 gene tom111. The expression of both genes was induced by high temperature in fruits, flowers, leaves, and stems, but not by low or ambient temperatures or by other stresses such as drought and anaerobic conditions. When the heated fruits were transferred to low temperature, tom66 and tom111 mRNA levels first decreased but were then reinduced. Induction was not observed in nonheated fruits at low temperature. Immunodetection of tom111-encoded protein indicated that this protein is present at low temperatures in the heated fruits. The results of this study show that the expression of tom66 and tom111 is correlated with protection against some, but not all, symptoms of chilling injury.

Dim-Red-Light-Induced Increase in Polar Auxin Transport in Cucumber Seedlings1 : I. Development of Altered Capacity, Velocity, and Response to Inhibitors

We have developed and characterized a system to analyze light effects on auxin transport independent of photosynthetic effects. Polar transport of [3H]indole-3-acetic acid through hypocotyl segments from etiolated cucumber (Cucumis sativus L.) seedlings was increased in seedlings grown in dim-red light (DRL) (0.5 μmol m−2 s−1) relative to seedlings grown in darkness. Both transport velocity and transport intensity (export rate) were increased by at least a factor of 2. Tissue formed in DRL completely acquired the higher transport capacity within 50 h, but tissue already differentiated in darkness acquired only a partial increase in transport capacity within 50 h of DRL, indicating a developmental window for light induction of commitment to changes in auxin transport. This light-induced change probably manifests itself by alteration of function of the auxin efflux carrier, as revealed using specific transport inhibitors. Relative to dark controls, DRL-grown seedlings were differentially less sensitive to two inhibitors of polar auxin transport, N-(naphth-1-yl) phthalamic acid and 2,3,5-triiodobenzoic acid. On the basis of these data, we propose that the auxin efflux carrier is a key target of light regulation during photomorphogenesis.

The Role of Iron-Deficiency Stress Responses in Stimulating Heavy-Metal Transport in Plants1

Plant accumulation of Fe and other metals can be enhanced under Fe deficiency. We investigated the influence of Fe status on heavy-metal and divalent-cation uptake in roots of pea (Pisum sativum L. cv Sparkle) seedlings using Cd2+ uptake as a model system. Radiotracer techniques were used to quantify unidirectional 109Cd influx into roots of Fe-deficient and Fe-sufficient pea seedlings. The concentration-dependent kinetics for 109Cd influx were graphically complex and nonsaturating but could be resolved into a linear component and a saturable component exhibiting Michaelis-Menten kinetics. We demonstrated that the linear component was apoplastically bound Cd2+ remaining in the root cell wall after desorption, whereas the saturable component was transporter-mediated Cd2+ influx across the root-cell plasma membrane. The Cd2+ transport system in roots of both Fe-deficient and Fe-sufficient seedlings exhibited similar Michaelis constant values, 1.5 and 0.6 μm, respectively, for saturable Cd2+ influx, whereas the maximum initial velocity for Cd2+ uptake in Fe-deficient seedlings was nearly 7-fold higher than that in Fe-grown seedlings. Investigations into the mechanistic basis for this response demonstrated that Fe-deficiency-induced stimulation of the plasma membrane H+-ATPase did not play a role in the enhanced Cd2+ uptake. Expression studies with the Fe2+ transporter cloned from Arabidopsis, IRT1, indicated that Fe deficiency induced the expression of this transporter, which might facilitate the transport of heavy-metal divalent cations such as Cd2+ and Zn2+, in addition to Fe2+.

Heat-Stress Response of Maize Mitochondria1

We have identified maize (Zea mays L. inbred B73) mitochondrial homologs of the Escherichia coli molecular chaperones DnaK (HSP70) and GroEL (cpn60) using two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblots. During heat stress (42°C for 4 h), levels of HSP70 and cpn60 proteins did not change significantly. In contrast, levels of two 22-kD proteins increased dramatically (HSP22). Monoclonal antibodies were developed to maize HSP70, cpn60, and HSP22. The monoclonal antibodies were characterized with regard to their cross-reactivity to chloroplastic, cytosolic, and mitochondrial fractions, and to different plant species. Expression of mitochondrial HSP22 was evaluated with regard to induction temperature, time required for induction, and time required for degradation upon relief of stress. Maximal HSP22 expression occurred in etiolated seedling mitochondria after 5 h of a +13°C heat stress. Upon relief of heat stress, the HSP22 proteins disappeared with a half-life of about 4 h and were undetectable after 21 h of recovery. Under continuous heat-stress conditions, the level of HSP22 remained high. A cDNA for maize mitochondrial HSP22 was cloned and extended to full length with sequences from an expressed sequence tag database. Sequence analysis indicated that HSP22 is a member of the plant small heat-shock protein superfamily.

Energy Sources for HCO3− and CO2 Transport in Air-Grown Cells of Synechococcus UTEX 6251

Light-dependent inorganic C (Ci) transport and accumulation in air-grown cells of Synechococcus UTEX 625 were examined with a mass spectrometer in the presence of inhibitors or artificial electron acceptors of photosynthesis in an attempt to drive CO2 or HCO3− uptake separately by the cyclic or linear electron transport chains. In the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, the cells were able to accumulate an intracellular Ci pool of 20 mm, even though CO2 fixation was completely inhibited, indicating that cyclic electron flow was involved in the Ci-concentrating mechanism. When 200 μm N,N-dimethyl-p-nitrosoaniline was used to drain electrons from ferredoxin, a similar Ci accumulation was observed, suggesting that linear electron flow could support the transport of Ci. When carbonic anhydrase was not present, initial CO2 uptake was greatly reduced and the extracellular [CO2] eventually increased to a level higher than equilibrium, strongly suggesting that CO2 transport was inhibited and that Ci accumulation was the result of active HCO3− transport. With 3-(3,4-dichlorophenyl)-1,1-dimethylurea-treated cells, Ci transport and accumulation were inhibited by inhibitors of CO2 transport, such as COS and Na2S, whereas Li+, an HCO3−-transport inhibitor, had little effect. In the presence of N,N-dimethyl-p-nitrosoaniline, Ci transport and accumulation were not inhibited by COS and Na2S but were inhibited by Li+. These results suggest that CO2 transport is supported by cyclic electron transport and that HCO3− transport is supported by linear electron transport.

Expression of the CD36 homolog (FAT) in fibroblast cells: effects on fatty acid transport.

An adipocyte membrane glycoprotein, (FAT), homologous to human CD36, has been previously implicated in the binding/transport of long-chain fatty acids. It bound reactive derivatives of long-chain fatty acids and binding was specific and associated with significant inhibition of fatty acid uptake. Tissue distribution of the protein and regulation of its expression were also consistent with its postulated role. In this report, we have examined the effects of FAT expression on rates and properties of fatty acid uptake by Ob17PY fibroblasts lacking the protein. Three clones (P21, P22, and P25) were selected based on FAT mRNA and protein levels. Cell surface labeling could be demonstrated with the anti-CD36 antibody FITC-OKM5. In line with this, the major fraction of immunoreactive FAT was associated with the plasma membrane fraction. Assays of oleate and/or palmitate uptake demonstrated higher rates in the three FAT-expressing clones, compared to cells transfected with the empty vector. Clone P21, which had the highest protein levels on Western blots, exhibited the largest increase in transport rates. Fatty acid uptake in FAT-expressing P21 cells reflected two components, a phloretin-sensitive high-affinity saturable component with a Km of 0.004 microM and a basal phloretin-insensitive component that was a linear function of unbound fatty acid. P21 cells incorporated more exogenous fatty acid into phospholipids, indicating that binding of fatty acids was followed by their transfer into the cell and that both processes were increased by FAT expression. The data support the interpretation that FAT/CD36 functions as a high-affinity membrane receptor/transporter for long-chain fatty acids.

Heat shock induces a loss of rRNA-encoding DNA repeats in Brassica nigra.

Stress-induced mutations may play an important role in the evolution of plants. Plants do not sequester a germ line, and thus any stress-induced mutations could be passed on to future generations. We report a study of the effects of heat shock on genomic components of Brassica nigra Brassicaceae. Plants were submitted to heat stress, and the copy number of two nuclear-encoded single-copy genes, rRNA-encoding DNA (rDNA) and a chloroplast DNA gene, was determined and compared to a nonstressed control group. We determined whether genomic changes were inherited by examining copy number in the selfed progeny of control and heat-treated individuals. No effects of heat shock on copy number of the single-copy nuclear genes or on chloroplast DNA are found. However, heat shock did cause a statistically significant reduction in rDNA copies inherited by the F1 generation. In addition, we propose a DNA damage-reppair hypothesis to explain the reduction in rDNA caused by heat shock.

Identification of C1q as the heat-labile serum cofactor required for immune complexes to stimulate endothelial expression of the adhesion molecules E-selectin and intercellular and vascular cell adhesion molecules 1.

To examine the role of complement components as regulators of the expression of endothelial adhesive molecules in response to immune complexes (ICs), we determined whether ICs stimulate both endothelial adhesiveness for leukocytes and expression of E-selectin and intercellular and vascular cell adhesion molecules 1 (ICAM-1 and VCAM-1). We found that ICs [bovine serum albumin (BSA)-anti-BSA] stimulated endothelial cell adhesiveness for added leukocytes in the presence of complement-sufficient normal human serum (NHS) but not in the presence of heat-inactivated serum (HIS) or in tissue culture medium alone. Depletion of complement component C3 or C8 from serum did not prevent enhanced endothelial adhesiveness stimulated by ICs. In contrast, depletion of complement component C1q markedly inhibited IC-stimulated endothelial adhesiveness for leukocytes. When the heat-labile complement component C1q was added to HIS, the capacity of ICs to stimulate endothelial adhesiveness for leukocytes was completely restored. Further evidence for the possible role of C1q in mediating the effect of ICs on endothelial cells was the discovery of the presence of the 100- to 126-kDa C1q-binding protein on the surface of endothelial cells (by cytofluorography) and of message for the 33-kDa C1q receptor in resting endothelial cells (by reverse transcription-PCR). Inhibition of protein synthesis by cycloheximide blocked endothelial adhesiveness for leukocytes stimulated by either interleukin 1 or ICs in the presence of NHS. After stimulation with ICs in the presence of NHS, endothelial cells expressed increased numbers of adhesion molecules (E-selectin, ICAM-1, and VCAM-1). Endothelial expression of adhesion molecules mediated, at least in part, endothelial adhesiveness for leukocytes, since leukocyte adhesion was blocked by monoclonal antibodies directed against E-selectin. These studies show that ICs stimulate endothelial cells to express adhesive proteins for leukocytes in the presence of a heat-labile serum factor. That factor appears to be C1q.

Endogenous mutagenesis by an insertion sequence element identifies Aeromonas salmonicida AbcA as an ATP-binding cassette transport protein required for biogenesis of smooth lipopolysaccharide.

Analysis of an Aeromonas salmonicida A layer-deficient/O polysaccharide-deficient mutant carrying a Tn5 insertion in the structural gene for A protein (vapA) showed that the abcA gene immediately downstream of vapA had been interrupted by the endogenous insertion sequence element ISAS1. Immunoelectron microscopy showed that O polysaccharides did not accumulate at the inner membrane-cytoplasm interface of this mutant. abcA encodes an unusual protein; it carries both an amino-terminal ATP-binding cassette (ABC) domain showing high sequence similarity to ABC proteins implicated in the transport of certain capsular and O polysaccharides and a carboxyl-terminal potential DNA-binding domain, which distinguishes AbcA from other polysaccharide transport proteins in structural and evolutionary terms. The smooth lipopolysaccharide phenotype was restored by complementation with abcA but not by abcA carrying site-directed mutations in the sequence encoding the ATP-binding site of the protein. The genetic organization of the A. salmonicida ABC polysaccharide system differs from other bacteria. abcA also differs in apparently being required for both O-polysaccharide synthesis and in energizing the transport of O polysaccharides to the cell surface.